A four-step regulatory cascade controls bistable transfer competence development of the integrative and conjugative element ICEclc in Pseudomonas

Genetic bistability controls different phenotypic programs in defined subpopulations of genetically identical bacteria. Conjugative transfer of the integrative and conjugative element ICE clc in Pseudomonas requires development of a transfer competence state in stationary phase, but this state arises only in 3-5% of individual cells. The mechanisms controlling and underlying the bistable switch between non-active and transfer competence cells have long remained enigmatic. Using a variety of genetic tools combined with stochastic modeling, we characterize here the factors and overall network architecture controlling bistable ICE clc activation of transfer competence. Two new key regulators (BisR and BisDC) were uncovered, that link the hierarchical cascade of ICE clc transfer competence activation to in total four regulatory nodes. The final activator complex named BisDC drives a positive feedback on its own transcription, and directly controls the “late” ICE promoters for excision and transfer. Stochastic mathematical modeling conceptually explained the arisal and maintenance of bistability by the feedback loop, and demonstrated its importance to guarantee consistent prolonged downstream output in activated cells. A minimized gene set allowing controllable bistable output in a Pseudomonas putida in absence of the ICE clc largely confirmed model predictions. Phylogenetic analyses further showed that the two new ICE clc regulatory factors are widespread among putative ICEs found in Gamma- and Beta -proteobacteria, highlighting the conceptual importance of our findings for the behaviour of this wide family of conjugative elements.